R0le of Complement in the Immunopathology of Macrophages

Newman, Simon

Abstract

The purpose of this research project is to investigate certain aspects of human monocyte differentiation and macrophage function that may be regulated by membrane complement (C) receptors or macrophage-endogenous C components. Purified complement components and F(ab')?2? and Fab' fragments of antibodies specific for either C components or C receptors are being used as probes of specific macrophage functions. Using these reagents, it has become clear that human in vitro monocytederived macrophages and human peritoneal macrophages possess structurally and functionally distinct receptors, CR?2? and CR?3?, that recognize the ligands C3b and iC3b, respectively; and that these two receptors dunction independently in promoting the phagocytosis of sheep erythrocytes (E) costed with C3b or iC3b. Research plans for the coming year have three main objectives: (1) to determine the role of macrophage CR?1? and CR?3? in promoting the phagocytosis and destruction of gram positive and gram negative microorganisms; (2) to examine macrophage release of lysosomal enzymes and toxic oxygen etabolites triggered by E and bacteria costed with different C components, in order to determine which C receptors or intrinsic C components are required either to trigger or modulate those reactions; and (3) to attempt to biochemically and physically characterize the mechanisms involved in C receptor-mediated phagocytosis. As has been done with E, bacteria will be coated with C3b using purified alternative complement pathway components, and converting bound C3b to iC3b with purified H and I. Microorganisms will be grown in media containing radiolabeled amino acids so that the extent of phagocytosis can be easily quantitated. Adherent but noningested bacteria will be removed from macrophage monolayers by trypsin or lysostaphin (for S. aureus). To determine phagocytic microbicidal activity, macrophage monolayers will be lysed with water, and bacterial viability determined by serial dilution and plating on nutrient agar. Macrophage release of lysosomal enzymes and the generation of the toxic oxygen metabolites superoxide anion (0?2?) and H?2?0?2? will be quantitated by standard biochemical techniques. In addition, superoxide dismutase and catalase will be added directly to bactericidal assays to determine the relative importance of 0?2? and H?2?0?2? in macrophage killing of microorganisms. Finally, various chemical agents will be added to macrophage monolayers during phagocytic assays to determine the contribution of microtubules, microfilaments, prostaglandins, and intracellular calcium to the phagocytic process. (MB)